1. Field of the Invention
The present invention relates to high purity polyethylene glycol derivatives useful as protein modifiers, etc., and a novel process for preparing polyethylene glycol derivatives as well as protein modified by high purity polyethylene glycol derivatives.
2. Related Art Statement
In recent years, it has become possible to produce protein having physiological activities in large quantities due to the progress of genetic engineering technology. Such protein has been expected to be used as a drug. However, when physiologically active protein is provided for practical use as a therapeutic agent, the protein is sometimes ineffective as a therapeutic agent, because its clearance from blood circulation is extremely rapid due to decomposition of the protein by peptidase present in the body, transfer of the protein to the target tissue is not efficient, etc.. Furthermore, there is a danger that immune reaction might be caused when physiologically active protein obtained from the heterologous organism is administered to human. In order to solve these problems, it has been attempted to chemically modify the physiologically active protein with an artificial high molecular compound, especially using polyethylene glycol. In polyethylene glycol, its immunogenicity per se is extremely low; by chemically binding polyethylene glycol to protein, the effects of decreasing antigenicity, decreasing immunogenicity, minimizing toxicity, prolonging plasma half life, etc. are exhibited.
In addition, the polyethylene glycol-bound protein is soluble in an organic solvent so that synthesis using hydrolase (i.e., protein) can be effectively conducted.
For chemically binding polyethylene glycol to protein, the following methods are known: (1) method for introducing two polyethylene glycol mono-alkyl ether chains into amino groups of protein via cyanuric chloride [Inada et al., Japanese Patent Application KOKOKU No. 61-42558; Inada et al., Chemistry Letters, 773 (1980); Inada et al., Japanese Journal of Cancer Research, 77, 1264 (1986); Miyata et al., Japanese Patent Application KOKAI No. 62-115280]; (2) method for introducing polyethylene glycol into amino groups of protein using polyethylene glycol mono-alkyl ether acyl azides (Theodous Fan, N., et al., Japanese Patent Application KOKOKU No. 56-23587); (3) method using polyethylene glycol mono-alkyl ether aldehydes (Fujino et al., Japanese Patent Application KOKAI No. 61-178926); (4) method for introducing polyethylene glycol mono-alkyl ethers into amino groups of protein via imidoyl groups (Fujino et al., Japanese Patent Application KOKAI No. 63-10800); (5) method using polyethylene glycol mono-alkyl ethers and N-hydroxysuccinimide [Leonard, M., et al., Tetrahedron, 40, 1581-1584 (1984) Abuchowski, A. et al., Cancer Biochem Biophys., 7, 175 (1984)]; (6) method for introducing single chain polyethylene glycol mono-alkyl ethers into amino groups of protein via cyanuric chloride [A. Abuchowski et al., J. Biol. Chem., 252., 3578 (1977)]; (7) method which comprises activating polyethylene glycol mono-alkyl ethers with carbonyldiimidazole and then introducing the activated compounds into amino groups of protein [Charles O.B. Cham, et al., Anal. Biochem., 131, 25 (1983)]; etc. Among these methods, the method (1) is concerned with modification method for introducing compounds represented by the following formula (I): ##STR1## [wherein R represents an alkyl group and n represents an optionally variable positive integer], which are derived from polyethylene glycol mono-alkyl ethers and cyanuric chloride, into amino groups and is characterized in that two polyethylene glycol chains can be introduced into one amino group, unlike other methods for modification (methods (2) through (7) described above). As modified protein according to this modification method, there are known asparaginase [Inada et al., Japanese Patent Application KOKOKU No. 61-42558; Inada et al., Chemistry Letters, 773 (1980); Inada et al., Japanese Journal of Cancer Research, 77, 1264 (1986)], superoxide dismutase Miyata et al., Japanese Patent Application KOKAI No. 2-115280]; and the like. Modified protein obtained using compounds represented by the following formula (III): ##STR2## [wherein R and n have the same significances as described above] was compared with modified protein obtained using compounds represented by formula (I). The comparison reveals that modification using compounds represented by formula (I) is more excellent in reducing the antigenicity and retaining the activity.
Now, a protein modifier having high purity is required for preparing modified protein. It was attempted to synthesize Compound (I) which is a protein modifier according to the method (1) recited in the publications supra. However, analysis by high performance gel filtration chromatography reveals that the reaction product was a mixture containing Compound (I) in any case. That is, in the attempt to obtain Compound (I) having a mean molecular weight of 10,000 using a polyethylene glycol mono-alkyl ether having a mean molecular weight of 5,000 and cyanuric chloride; (a) according to the method described in Japanese Patent Application KOKOKU No. 61-42558, 20 g of monomethoxypolyethylene glycol having a molecular weight of 5,000 was dissolved in 100 ml of anhydrous benzene containing 10 g of anhydrous sodium carbonate, the solution was refluxed at 80.degree. C. for 30 minutes, 365 mg of 2,4,6-trichloro-s-triazine was then added to the reaction mixture to react them while refluxing at 80.degree. C. for 24 hours, the reaction residue was filtered off, 300 ml of petroleum ether was added to cause precipitation and the precipitates were washed with petroleum ether several times. Further (b) according to the method described in Japanese Patent Application KOKAI No. 62-115280, 730 mg of cyanuric chloride was added to a mixture of 40 g of polyethylene glycol monomethyl ether (having a mean molecular weight of 5,000), 200 ml of benzene, 20 g of anhydrous sodium carbonate and 10 g of molecular sieve 3A; the resulting mixture was reacted at 80.degree. C. for 20 hours, and then the procedures of adding 400 ml of petroleum ether to the reaction mixture to cause precipitation, dissolving the precipitates in benzene and precipitating again with petroleum ether were repeated 3 times. In both of the methods (a) and (b), there was obtained a mixture of several compounds including Compound (III) (R=CH.sub.3) as the main product which have molecular weights over a wide range of from 5,000 to high molecular region (FIGS. 2 and 3). Furthermore, according to the method described in Chemistry Letters, 773 (1980), 730 mg of cyanuric chloride was added to a mixture of 40 g of polyethylene glycol monomethyl ether (a mean molecular weight of 5,000), 200 ml of benzene, 20 g of anhydrous sodium carbonate and 10 g of molecular sieve 3A; the resulting mixture was reacted at 80.degree. C. for 44 hours, and the procedures of precipitating with 400 ml of petroleum ether, dissolving the precipitates in benzene and precipitating again with petroleum ether were repeated 6 times to obtain a mixture of Compound (III) (R=CH.sub.3), Compound (I) (R=CH.sub.3) and compounds possessing higher molecular weight (FIG. 4). Still further according to the method described in Japanese Journal of Cancer Research, 77, 1264 (1986), 1.12 g of cyanuric chloride was added to a mixture of 60 g of polyethylene glycol monomethyl ether, 200 ml of anhydrous benzene, 20 g of anhydrous sodium carbonate and 20 g of molecular sieve 4A; the resulting mixture was reacted at 80.degree. C. for 120 hours, benzene was distilled off, and then the procedures of dissolving the residue in acetone and precipitating with petroleum ether were repeated 3 times to obtain a mixture of various compounds which mainly contained the products with higher molecular weight (FIG. 5). It is also mentioned in this journal that gel filtration chromatography was carried out on Sephadex G-100 as a carrier to obtain the pure product showing a single peak, which is corresponded to the molecular weight of 10,000, in this chromatography, indicating that homogeneous Compound (I) (R=CH.sub.3) was obtained. However, high performance gel filtration chromatography having an excellent separation ability as compared to low speed gel filtration chromatography using Sephadex G-100 or the like as a carrier has been recently developed and as the result, separation which was impossible in the past became possible [Seikagaku, 56, 1481 (1984)]. That is, analysis by means of low speed gel filtration chromatography using Sephadex G-100 or the like as a carrier is insufficient for analysis of purity. In fact, according to the method described in this journal, gel filtration chromatography was performed on Sephadex G-100, and the resulting part, which was the main peak in this chromatography, corresponded to the peak showing a molecular weight of 10,000 in this journal and showed a single peak on gel filtration chromatography using Sephadex G-100 as carrier, was further analyzed by high performance gel filtration chromatography. The result showed that this part was a mixture of various compounds with the major compounds being those with higher molecular weight (FIG. 6).
It is the actual situation that it has been unsuccessful so far to obtain the desired Compound (I) efficiently from such a mixture containing various compounds having molecular weights over a wide range by means of industrial separation and purification (recrystallization, reprecipitation, ultrafiltration, etc.).
On the other hand, where protein is modified using such a mixture containing various compounds having molecular weights over a wide range, the modified protein is not uniform in quality. It is thus extremely difficult to obtain the product having constant quality. In case that such protein is used as a therapeutic agent, various problems such as side effects, etc. might be caused due to impurities.